The increase in antiretroviral use globally to treat HIV infections has been a much needed and worthy achievement as many thousands of lives have been saved by the rapid expansion of treatment. A natural byproduct of the increased use of these medications is the acquisition of drug resistance in patients failing therapy that will ultimately result in transmitted drug resistance. The timing of the increase in transmitted resistance so that it impacts national antiretroviral therapy (ART) programs is unclear, but in resource-limited regions, where it is not feasible to test all individuals for drug resistance at the time they enter care, alternative strategies to monitor resistance are needed. We hypothesize that transmitted drug resistance can be monitored with novel strategies in Botswana and that characterizing the viral fitness of HIV-1 subtype C with resistance mutations to 1st line antiretroviral therapy will assist in predicting the rate of rise in transmitted resistance. In Specific Aim 1, we propose to conduct surveillance for transmitted HIV drug resistance in Botswana for four consecutive years using a surveillance method developed by the World Health Organization (WHO) in addition to our novel strategy of pooling samples and applying highly sensitive assays to detect drug resistance mutations critical to 1st line ART. This strategy will be validated by the standard genotyping results obtained by the WHO protocol. In Specific Aim 2, this method of pooling samples will be compared to a standard in-house genotyping technique to determine if this approach to genotyping provides meaningful results prior to ART initiation. The data generated by this technique would again focus on those mutations relevant to 1st line ART to aid clinicians in predicting success of this therapy. The viral fitness of drug resistant viruses will be the focus of Specific Aim 3, as fitness is a key determinant in the transmission of drug resistant virus. The recent change in 1st line ART in Botswana from zidovudine to tenofovir-based therapy may produce viruses with different fitness in individuals who fail therapy. This will be examined in vitro as mutations in HIV-1 subtype C that develop with tenofovir-based ART possibly occur more frequently in this population, and if these viruses have greater relative fitness, they are more capable of forward transmission. The studies to be performed will characterize the current state of transmitted drug resistance in Botswana by conventional and novel techniques, determine the feasibility of new strategies to monitor for the emergence of HIV drug resistance in resource-limited regions, and further our understanding of the viral characteristics of HIV-1 subtype C as drug resistance develops;all essential components to the continued success of the ART program in Botswana. PUBLIC HEALTH RELEVANCE: Transmitted HIV drug resistance will undoubtedly increase in resource-limited regions as antiretroviral therapy becomes more available and this resistance will impact the success of national antiretroviral programs. The goals of this proposal are to 1) evaluate the extent of transmitted drug resistance in Botswana using the WHO recommended surveillance method in addition to a novel, less expensive approach that identifies those resistance mutations critical to 1st line antiretroviral therapy, to 2) assess if similar techniques can serve as a low cost alternative to genotyping prior to initiation of antiretroviral treatment, and 3) characterize the in vitro fitness of resistant viruses that arise with 1st line therapy as viral fitness is a core determinant of transmitted drug resistance. These results will expand our knowledge about the current state of drug resistance in Botswana and provide in vitro data regarding the fitness of viral mutations that will determine the trajectory of transmitted drug resistance.